Two new species of

Two new species of Chalciporus (Boletaceae, Boletales), viz. C. sinensis and C. vulparius, are described based on morphological and molecular data. Detailed descriptions, color photographs of fresh basidiomata, and line drawings of microscopic features of the two new taxa are presented. In addition, a key to all known species of Chalciporus from China is provided.

Hainan Medical University (FHMU), Haikou City, Hainan Province, China. Color codes are based on Kornerup and Wanscher (1981). Sections of the pileipellis were cut longitudinally and halfway between the center and margin of the pileus. Sections of the stipitipellis were obtained from the middle part along the longitudinal axis of the stipe. Samples were rehydrated with 5% KOH and mounted on microscope slides. All microscopic structures were drawn freehand. The number of basidiospores is given as n/m/p, i.e., n basidiospores from m basidiomata from p collections were counted. The basidiospore dimensions are given as (a)b-c(d), where b and c represent the 5th and 95th percentile, respectively, and extreme values are shown in parentheses. Q refers to the length/width ratio of basidiospores, and Qm is the average Q and standard deviation. The terms related to the size of basidioma are based on the study by Bas (1969).

Molecular procedures
Total genomic DNA was obtained using the Plant Genomic DNA Kit (KANGWEI Company, China) according to the manufacturer's instructions from material dried in silica gel. The primer pairs used for amplification were LR0R/LR5 (Vilgalys and Hester 1990;James et al. 2006) for the nuc 28S rDNA D1-D2 domains (28S), ITS5/ITS4 (White et al. 1990) for the nuc rDNA region encompassing the internal transcribed spacers 1 and 2 and for 5.8S rDNA (ITS), EF1-2F/EF1-2R (Zeng et al. 2013) for the translation elongation factor 1-α gene (TEF1), and RPB2-B-R/RPB2-B-F1 (Wu et al. 2014) for the gene for the RNA polymerase II second largest (RPB2) subunit. PCR was performed in a reaction volume of 25 μl containing 13 μl of 2 × Taq PCR MasterMix (KANGWEI Company, China), 2 μl of each primer (10 μM), 8 μl of nuclease-free water, and 2 μl of DNA template. Amplification reactions included one cycle of denaturation at 95 °C for 4 min, followed by 34 cycles of denaturation at 94 ℃ for 30 s, annealing at the appropriate temperature (50 ℃ for 28S and ITS, 53 ℃ for TEF1, and 52 ℃ for RPB2) for 30 s, extension at 72 ℃ for 120 s, and one extension cycle at 72 ℃ for 7 min. PCR products were separated in 1% (w/v) agarose gels, and amplicons were purified and sequenced using an ABI 3730xl DNA Analyzer (Guangzhou Branch of BGI, China) with the same primers used in PCR. Forward or reverse sequences were compiled with BioEdit (Hall 1999).

Dataset assembly
Twenty sequences (six of 28S, four of ITS, six of TEF1, and four of RPB2) from six collections were generated, and edited sequences were deposited in GenBank. The Gen-Bank accession numbers of ITS sequences are MW917178 to MW917181, and those of 28S, TEF1, and RPB2 are listed in Table 1. 28S, TEF1, and RPB2 sequences were aligned with sequences obtained from GenBank and previous studies (Table 1). Buchwaldoboletus xylophilus (Petch) Both & B. Ortiz was chosen as the outgroup based on phylogeny (Xie et al. 2021). Single-locus phylogenetic trees based on 28S, TEF1 (exons only), and RPB2 (exons only) sequences were aligned using MUSCLE (Edgar 2004). There were no conflicts in the topologies of these trees, indicating that phylogenetic signals from different gene fragments were congruent. Then sequences were concatenated using Phyutility version 2.2 (Smith and Dunn 2008).

Phylogenetic analyses
The combined nuclear dataset (28S + TEF1 + RPB2) was analyzed using maximum likelihood (ML) and Bayesian inference (BI) methods. The ML tree was generated, and bootstrap analyses were performed using RAxML version 7.2.6 (Stamatakis 2006). ML searches were performed using RAxML 7.2.6 program and involved 1000 repetitions under the GTRGAMMA model, and all model parameters were estimated by this software. BI was performed using the Markov Chain Monte Carlo technique in MrBayes version 3.1 (Huelsenbeck and Ronquist 2005), and parameters were predetermined using MrModeltest version 2.3 (Nylander 2004). The evolution model used in BI was determined with MrModeltest 2.3 (Nylander 2004). For the combined dataset, GTR + I + G, GTR + I + G, and SYM + G were chosen as the best-fit likelihood models for 28S, TEF1, and RPB2, respectively. Bayesian analysis of the combined dataset (28S + TEF1 + RPB2) was repeated for 0.4 million generations and sampled every 100th generation. Trees sampled from the first 25% of the generations were discarded as burnin, and Bayesian posterior probabilities (PP) were calculated for the majority consensus of the retained Bayesian trees.

Results
The combined Chalciporus dataset (28S + TEF1 + RPB2) consisted of 33 taxa and 2159 nucleotide sites, and alignments were deposited in TreeBASE (http:// purl. org/ phylo/ treeb ase/ phylo ws/ study/ TB2: S28091). Phylogenetic trees based on the combined dataset generated from ML and BI analyses were congruent except for slight differences of statistical support. The phylogram generated from RAxML, including support values, is shown in Fig. 1.
Habitat: Scattered on the ground in forests dominated by fagaceous trees.
Phylogenetically, C. vulparius is closely related to C. africanus and C. hainanensis and C. sinensis (Fig. 1). However, C. africanus has a reddish brown, grayish orange, cinnamon to cognac-brown stipe, a yellow basal mycelium, and a large value of Qm, and it is distributed in Africa (Degreef and Kesel 2008). The morphological differences between C. vulparius and the two Chinese taxa, viz. C. sinensis and C. hainanensis, were discussed above.

Discussion
Molecular data indicate that the newly collected specimens were nested into the genus Chalciporus with high statistical support (Fig. 1), and morphological features of the two new species, viz. C. sinensis and C. vulparius, are consistent with those of Chalciporus.
The present study showed high species diversity in China and identified eight lineages of Chalciporus (Fig. 1). Lineages 7 and 8 (C. vulparius and C. sinensis) are new; lineages 1, 2, 3, and 5 (C. citrinoaurantius, C. rubinelloides, C. radiatus, and C. hainanensis) were described previously, and lineages 4 and 6 were not described because of the paucity of material. Chalciporus piperatus was described previously (Li and Song, 2002); however, its occurrence in China needs to be confirmed.
The present data indicate that the species of Chalciporus in China are distributed in subtropical and tropical areas, and no species of this genus were identified in temperate regions of the country (Fig. 1). With more field investigations, more tropical Chalciporus species are expected to be discovered in China. Moreover, we noted that the affinities of Chalciporus species from East Asia and southeast Asia are evident (Fig. 1), and both regions shared one common taxon (lineage 5, Fig. 1). Similar scenarios have been documented for many other boletes (Zeng et al. 2013(Zeng et al. , 2018Wu et al. 2019).